Astrocyte-endothelial crosstalk after cerebral ischemia and hemorrhage Reactive astrocytes were traditionally thought to inhibit neuronal plasticity after CNS injury. But emerging data now suggest that reactive astrocytes may also have beneficial actions. Our pilot data suggest that (i) reactive astrocytes release HMGB1 that can promote angiogenesis, (ii) downregulating the release of HMGB1 from reactive astrocytes may worsen neurovascular recovery after focal ischemia in mice, (iii) HMGB1 may upregulate RAGE receptors on cerebral endothelial cells, (iv) increased endothelial RAGE may enhance targeted adhesion of endothelial progenitor cells via beta-2 integrins, and (v) HMGB1 may increase proliferation, maturation and angiogenesis in endothelial progenitor cells, thus promoting repair after stroke. Based on these pilot data, we hypothesize that astrocyte-endothelial crosstalk is essential for neurovascular recovery after stroke: reactive astrocytes release HMGB1 that upregulates RAGE receptor on cerebral endothelium;RAGE binds beta-2 integrins on circulating endothelial progenitor cells thus pulling them into recovering brain;and once endothelial progenitors arrive, HMGB1 promotes their proliferation, maturation and angiogenesis. Importantly, we propose that this pathway can promote recovery after both ischemic or hemorrhagic strokes. We will test this hypothesis in three aims. In Aim 1, we ask how HMGB1 from stimulated astrocytes upregulate RAGE on cerebral endothelial cells and enhance the targeted adhesion of endothelial progenitor cells. In Aim 2, we dissect mechanisms that underlie the ability of HMGB1 to enhance proliferation, maturation and angiogenesis in endothelial progenitor cells. In Aim 3, we will use mouse models of focal cerebral ischemia and intracerebral hemorrhage to confirm these astrocyte-endothelium-EPC mechanisms and show that they actually mediate neurovascular recovery in vivo. To test our pathways, we will use a combination of cell culture, in vivo mouse models, pharmacologic inhibitors, molecular techniques including siRNA, long-term neurological outcomes, and in vivo imaging. This study should define a novel mechanism wherein crosstalk between reactive astrocytes, cerebral endothelium, and circulating endothelial progenitor cells underlie neurovascular recovery after cerebral ischemia and hemorrhage. Dissecting these cell-cell signaling pathways may lead to new therapeutic approaches for promoting functional recovery in patients after ischemic and hemorrhagic strokes. PUBLIC HEALTH RELEVANCE: For R01 application by Eng H. Lo Astrocyte-endothelial crosstalk after cerebral ischemia and hemorrhage An important direction for stroke research involves the investigation of new therapies to improve recovery. However, the underlying mechanisms of how brain responds to and repairs after stroke remain unclear. Here, we will define a new mechanism that involves novel signaling between brain astrocytes, endothelium and endothelial progenitor cells. Our findings may be eventually used to develop new therapies for improving recovery after both ischemic and hemorrhagic strokes.